Proteomic Mapping of ER-PM Junctions Identifies STIMATE as a Regulator of Ca²⁺ Influx

Nat Cell Biol. 2015 Oct;17(10):1339-47. doi: 10.1038/ncb3234. Epub 2015 Aug 31.

Abstract

Specialized junctional sites that connect the plasma membrane (PM) and endoplasmic reticulum (ER) play critical roles in controlling lipid metabolism and Ca(2+) signalling. Store-operated Ca(2+) entry mediated by dynamic STIM1-ORAI1 coupling represents a classical molecular event occurring at ER-PM junctions, but the protein composition and how previously unrecognized protein regulators facilitate this process remain ill-defined. Using a combination of spatially restricted biotin labelling in situ coupled with mass spectrometry and a secondary screen based on bimolecular fluorescence complementation, we mapped the proteome of intact ER-PM junctions in living cells without disrupting their architectural integrity. Our approaches led to the discovery of an ER-resident multi-transmembrane protein that we call STIMATE (STIM-activating enhancer, encoded by TMEM110) as a positive regulator of Ca(2+) influx in vertebrates. STIMATE physically interacts with STIM1 to promote STIM1 conformational switch. Genetic depletion of STIMATE substantially reduces STIM1 puncta formation at ER-PM junctions and suppresses the Ca(2+)-NFAT signalling. Our findings enable further genetic studies to elucidate the function of STIMATE in normal physiology and disease, and set the stage to uncover more uncharted functions of hitherto underexplored ER-PM junctions.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Biological Transport
  • COS Cells
  • Calcium / metabolism*
  • Cell Membrane / metabolism*
  • Chlorocebus aethiops
  • Endoplasmic Reticulum / metabolism*
  • Gene Expression Profiling
  • Gene Silencing
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Microscopy, Confocal
  • Microscopy, Fluorescence / methods
  • Molecular Sequence Data
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / metabolism
  • Proteome / metabolism*
  • Proteomics / methods
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Homology, Amino Acid
  • Stromal Interaction Molecule 1

Substances

  • Membrane Proteins
  • Neoplasm Proteins
  • Proteome
  • STIM1 protein, human
  • STIMATE protein, human
  • Stromal Interaction Molecule 1
  • Calcium